Feedback regulation of pulsatile Erk activity
Alexander G. Goglia1,2, Maxwell Z. Wilson1, Jared E. Toettcher1*
1Princeton University, Department of Molecular Biology; 2Rutgers Robert Wood Johnson Medical School
The Ras/Erk mitogen activated protein kinase (MAPK) pathway regulates many processes including proliferation, migration, and survival. Receptor-level input signals to Ras are transmitted in single cells as short pulses of Erk kinase activity, and the dynamics of Erk activity are thought to allow this pathway to control multiple divergent cell fate decisions. Though the network topology of Ras/Erk signaling has been the subject of intense study, how complex Erk dynamics arise is unclear. Here, we combine optogenetic control of Ras/Erk signaling with highly sensitive live-cell reporters to investigate this question. We show that a single cell can exhibit pulsatile dynamics of Erk kinase activity (visualized using the ErkKTR biosensor) but not Erk localization. Turning the Ras/Erk pathway on and off with our optogenetic Ras system (termed OptoSOS) results in immediate localization changes of both reporters, suggesting that the dynamic regulation of Erk activity is uncoupled from the dynamics of upstream pathway activity. Supporting this hypothesis, we found that continuous light stimulation of OptoSOS induces high, constant levels of cytoplasmic phospho-Erk that is presumably driven by constant upstream MEK activity, whereas nuclear phospho-Erk adapts back to baseline levels on a timescale consistent with observed pulsing. We next examined whether activation of the putative negative regulator requires new protein synthesis by globally inhibiting both transcription and translation. Although translation inhibitors lead to acute Erk activation, we find that pulsing is unaffected by the inhibition of transcription, indicating that new transcript synthesis is not required for Erk dynamics. These findings indicate that Erk is the direct target of transcription-independent negative feedback, which determines its endogenous pulsatile dynamics.